In this study, the procedure of SRAP - PCR was determined ; an initial denaturing step was performed at 94 X for 5 min; followed by 5 cycles at 94 X for 1 min, 33 X for 1 min and 72 X for 1 min; followed by 35 cycles at 94 X for 1 min, 50 X for 1 min, 72 X for 1 min, and a final extension at 72 X for 10 min. The orthogonal design was used to optimize SRAP-PCR system with five factors at three levels respectively based on capillary electrophoresis. The results showed that the optimized SRAP reaction mixtures for Paeonia suffruticosa Andr. ; (total volume 25 jxL) 50 ng DNA template, 0. 25 mmolL dNTP, 2. 5 mmol/L Mg + , 0.4 (xmol/Lprimer, 0. 5 U Taq DNA polymerase. The concentration of dNTP had the greatest effect and DNA template had the least effect on the result. And twenty-six pairs of primers were selected with abundant polymorphism above 70% from 756 pairs of primers. The optimized SRAP-PCR system and pairs of primers could be applied to related research of Paeonia suffruticosa Andr.%以牡丹叶片DNA为模板,对SRAP -PCR反应程序进行研究,确定适合的反应程序,即94℃预变性5 min; 94℃变性1 min,33℃退火1 min,72℃延伸1 min,共5个循环；随后94℃变性1 min,52℃退火1 min,72℃延伸1 min,共35个循环；最后72℃延伸10 min.基于毛细管电泳技术,采用正交设计L18( 37)对牡丹SRAP - PCR反应体系的5因素(Taq酶,Mg2+,模板DNA,dNTP,引物)3个水平进行了优化,构建了牡丹SRAP最佳反应体系:模板DNA 50 ng,dNTP 0.25 mmol/L,Mg2浓度2.5 mmol/L,引物浓度0.4 μmol/L,TaqDNA聚合酶0.5U,总体积为25 μL.各因素对扩增结果影响程度均不同:dNTPs＞引物＞DNA模板＞Mg2+＞Taq酶.运用该体系从756个SRAP引物组合中筛选出多态性好、条带清晰的26个引物组合,并证明了该体系稳定可靠.该体系的建立以及引物组合的确定为今后利用SRAP分子技术进行牡丹的相关研究奠定了科学基础.
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